TGF BETA INDUCED DECREASES OF ENDOTHELIAL INTEGRITY

点击翻译按钮获取中文摘要

Increases in pericellular transport due to changes in endothelial cell shape and the formation of intercellular gaps has been implicated as the major pathway for increased solute flux in a number of disease states including vascular remodeling found during pulmonary hypertension. Endothelial cell shape and gap formation is believed to be controlled by signal transduction pathways that alter the balance of competing adhesive and contractile forces. The experiments outlined in this grant will investigate if decreases in cell-cell adhesion, which tethers endothelial cells to one another, contributes to the formation of intercellular gaps following the addition of the Cytokine transforming growth factor beta, (TGF-beta). TGF-beta is a cytokine which is known alter endothelial cell shape in confluent monolayers and concomitantly increases the permeability. I hypothesize the following mechanism to explain the TGF- beta induced shape change and the formation of intercellular gaps. TGF- beta will promote cell separation by decreasing Ca++ dependent cell-cell adhesion. This decrease in Ca++ dependent cell-cell adhesion, which is believed to mediated by cadherins, will be the result of a decrease in the affinity of cadherin homophilic binding and not the result of a decrease in cadherin surface expression. Then, after cell separation has occurred, the adherens junction disassembles. Moreover, while the TGF-beta induced change in cell shape is dependent on basal centripetal tension, TGF-beta will not increase endothelial cell contractile activity as has been demonstrated for mediators such as thrombin. This hypothesis will be tested by completing the following specific aims: 1) To determine if Ca++ dependent or Ca++ independent cell-cell adhesion is decreased in endothelial cells by TGF-beta. Changes in the expression of cadherins, which are believed to mediate Ca++ dependent cell adhesion will also be determined following treatment with TGF-beta. 2) To determine the kinetics of adherens junction disassembly following exposure of endothelial cell monolayers to TGF-beta. 3) To determine if phosphorylation of proteins in the adherens junction by tyrosine kinases are part of the signal transduction pathway in TGF-beta induced increases in endothelial monolayer permeability. 4) To determine the contribution of both changes in endothelial cell contractile state as well as reorganization of the actin cytoskeleton to TGF-beta mediated changes in cell shape.